1. Field of the Invention
This invention relates to processes and compositions involved in dicarboxylic acid production in yeast. More particularly, the invention relates to a novel gene which encodes a cytochrome b5 protein in Candida tropicalis. 
2. Description of Related Art
Aliphatic dioic acids are versatile chemical intermediates useful as raw materials for the preparation of perfumes, polymers, adhesives and macrolid antibiotics. While several chemical routes to the synthesis of long-chain xcex1, xcfx89-dicarboxylic acids are available, the synthesis is not easy and most methods result in mixtures containing shorter chain lengths. As a result, extensive purification steps are necessary. While it is known that long-chain dioic acids can also be produced by microbial transformation of alkanes, fatty acids or esters thereof, chemical synthesis has remained the most commercially viable route, due to limitations with the current biological approaches.
Several strains of yeast are known to excrete xcex1, xcfx89-dicarboxylic acids as a byproduct when cultured on alkanes or fatty acids as the carbon source. In particular, yeast belonging to the Genus Candida, such as C. albicans, C. cloacae, C. guillermondii, C. intermedia, C. lipolytica, C. maltosa, C. parapsilosis and C. zeylenoides are known to produce such dicarboxylic acids (Agr. Biol. Chem. 35: 2033-2042 (1971)). Also, various strains of C. tropicalis are known to produce dicarboxylic acids ranging in chain lengths from C11 through C18 (Okino et al., B M Lawrence, B D Mookherjee and B J Willis (eds), in Flavors and Fragrances: A World Perspective. Proceedings of the 10th International Conference of Essential Oils, Flavors and Fragrances, Elsevier Science Publishers BV Amsterdam (1988)), and are the basis of several patents as reviewed by Bxc3xchler and Schindler, in Aliphatic Hydrocarbons in Biotechnology, H. J. Rehm and G. Reed (eds), Vol. 169, Verlag Chemie, Weinheim (1984).
Studies of the biochemical processes by which yeasts metabolize alkanes and fatty acids have revealed three types of oxidation reactions: xcex1-oxidation of alkanes to alcohols, (xcfx89-oxidation of fatty acids to xcex1, xcfx89-dicarboxylic acids and the degradative xcex2-oxidation of fatty acids to CO2 and water. The first two types of oxidations are catalyzed by microsomal enzymes while the last type takes place in the peroxisomes. In C. tropicalis, the first step in the xcfx89-oxidation pathway is catalyzed by a membrane-bound enzyme complex (xcfx89-hydroxylase complex) including a cytochrome P450 monooxygenase and a NADPH cytochrome reductase. This hydroxylase complex is responsible for the primary oxidation of the terminal methyl group in alkanes and fatty acids as described, e.g., in Gilewicz et al., Can. J. Microbiol. 25:201 (1979), incorporated herein by reference. The genes which encode the cytochrome P450 and NADPH reductase components of the complex have previously been identified as P450ALK and P450RED respectively, and have also been cloned and sequenced as described, e.g., in Sanglard et al., Gene 76:121-136 (1989), incorporated herein by reference. P450ALK has also been designated P450ALK1. More recently, ALK genes have been designated by the symbol CYP and RED genes have been designated by the symbol CPR. See, e.g., Nelson, Pharmacogenetics 6(1):1-42 (1996), which is incorporated herein by reference. See also Ohkuma et al., DNA and Cell Biology 14:163-173 (1995), Seghezzi et al., DNA and Cell Biology, 11:767-780 (1992) and Kargel et al., Yeast 12:333-348 (1996), each incorporated herein by reference. For example, P450ALK is also designated CYP52 according to the nomenclature of Nelson, supra. Fatty acids are ultimately formed from alkanes after two additional oxidation steps, catalyzed by alcohol oxidase as described, e.g., in Kemp et al., Appl. Microbiol. and Biotechnol. 28: 370-374 (1988), incorporated herein by reference, and aldehyde dehydrogenase. The fatty acids can be further oxidized through the same or similar pathway to the corresponding dicarboxylic acid. The xcfx89-oxidation of fatty acids proceeds via the xcfx89-hydroxy fatty acid and its aldehyde derivative, to the corresponding dicarboxylic acid without the requirement for CoA activation. However, both fatty acids and dicarboxylic acids can be degraded, after activation to the corresponding acyl-CoA ester through the xcex2-oxidation pathway in the peroxisomes, leading to chain shortening. In mammalian systems, both fatty acid and dicarboxylic acid products of xcfx89-oxidation are activated to their CoA-esters at equal rates and are substrates for both mitochondrial and peroxisomal xcex2-oxidation (J. Biochem., 102:225-234 (1987)). In yeast, xcex2-oxidation takes place solely in the peroxisomes (Agr. Biol. Chem. 49:1821-1828 (1985)).
The production of dicarboxylic acids by fermentation of unsaturated C14-C16 monocarboxylic acids using a strain of the species C. tropicalis is disclosed in U.S. Pat. No. 4,474,882. The unsaturated dicarboxylic acids correspond to the starting materials in the number and position of the double bonds. Similar processes in which other special microorganisms are used are described in U.S. Pat. Nos. 3,975,234 and 4,339,536, in British Patent Specification 1,405,026 and in German Patent Publications 21 64 626, 28 53 847, 29 37 292, 29 51 177, and 21 40 133.
Cytochrome P450 monooxygenases (P450s) are terminal monooxidases of a multicomponent enzyme system including P450 and CPR. In some instances, a second electron carrier, cytochrome b5(CYTb5) and its associated reductase are involved as described below and in Morgan, et al., Drug Metab. Disp. 12:358-364, 1984. The P450s comprise a superfamily of proteins which exist widely in nature having been isolated from a variety of organisms as described e.g., in Nelson, supra. These organisms include various mammals, fish, invertebrates, plants, mollusk, crustaceans, lower eukaryotes and bacteria (Nelson, supra). First discovered in rodent liver microsomes as a carbon-monoxide binding pigment as described, e.g., in Garfinkel, Arch. Biochem. Biophys. 77:493-509 (1958), which is incorporated herein by reference, P450s were later named based on their absorption at 450 nm in a reduced-CO coupled difference spectrum as described, e.g., in Omura et al., J. Biol. Chem. 239:2370-2378 (1964), which is incorporated herein by reference.
P450s catalyze the metabolism of a variety of endogenous and exogenous compounds as described, e.g., in Nelson, supra, and Nebert et al., DNA Cell. Biol. 10:1-14 (1991), which is incorporated herein by reference. Endogenous compounds include steroids, prostanoids, eicosanoids, fat-soluble vitamins, fatty acids, mammalian alkaloids, leukotrines, biogenic amines and phytolexins (Nelson, supra, and Nebert et al., supra). P450 metabolism involves such reactions as epoxidation, hydroxylation, dealkylation, hydroxylation, sulfoxidation, desulfuration and reductive dehalogenation. These reactions generally make the compound more water soluble, which is conducive for excretion, and more electrophilic. These electrophilic products can have detrimental effects if they react with DNA or other cellular constituents. However, they can react through conjugation with low molecular weight hydrophilic substances resulting in glucoronidation, sulfation, acetylation, amino acid conjugation or glutathione conjugation typically leading to inactivation and elimination as described, e.g., in Klaassen et al., Toxicology, 3rd ed, Macmillan, New York, 1986, incorporated herein by reference.
P450s are heme thiolate proteins consisting of a heme moiety bound to a single polypeptide chain of 45,000 to 55,000 Da. The iron of the heme prosthetic group is located at the center of a protoporphyrin ring. Four ligands of the heme iron can be attributed to the porphyrin ring. The fifth ligand is a thiolate anion from a cysteinyl residue of the polypeptide. The sixth ligand is probably a hydroxyl group from an amino acid residue, or a moiety with a similar field strength such as a water molecule as described, e.g., in Goeptar et al., Critical Reviews in Toxicology 25(1):25-65 (1995), incorporated herein by reference.
Monooxygenation reactions catalyzed by cytochromes P450 in a eukaryotic membrane-bound system require the transfer of electrons from NADPH to P450 via NADPH-cytochrome P450 reductase (CPR) as described, e.g., in Taniguchi et al., Arch. Biochem. Biophys. 232:585 (1984), incorporated herein by reference. CPR genes are now also referred to as NCP genes. See, e.g., Debacker et al., Antimicrobial Agents and Chemotherapy, 45:1660 (2001). CPR is a flavoprotein of approximately 78,000 Da containing 1 mol of flavin adenine dinucleotide (FAD) and 1 mol of flavin mononucleotide (FMN) per mole of enzyme as described, e.g., in Potter et al., J. Biol. Chem. 258:6906 (1983), incorporated herein by reference. The FAD moiety of CPR is the site of electron entry into the enzyme, whereas FMN is the electron-donating site to P450 as described, e.g., in Vermilion et al., J. Biol. Chem. 253:8812 (1978), incorporated herein by reference. The overall reaction is as follows:
H++RH+NADPH+O2xe2x86x92ROH+NADP++H2O
Binding of a substrate to the catalytic site of P450 apparently results in a conformational change initiating electron transfer from CPR to P450. Subsequent to the transfer of the first electron, O2 binds to the Fe2+-P450 substrate complex to form Fe3+-P450-substrate complex. This complex is then reduced by a second electron from CPR, or, in some cases, NADH via a second electron carrier, cytochrome b5 (CYTb5) and its associated NADH-cytochrome b5 reductase as described, e.g., in Guengerich et al., Arch. Biochem. Biophys. 205:365 (1980), incorporated herein by reference, and Morgan, supra. Most of the aforementioned studies implicate CYTb5 as being involved in the pathway only for the transfer of the second electron. One atom of this reactive oxygen is introduced into the substrate, while the other is reduced to water. The oxygenated substrate then dissociates, regenerating the oxidized form of the cytochrome P450 as described, e.g., in Klassen, Amdur and Doull, Casarett and Doull""s Toxicology, Macmillan, New York (1986), incorporated herein by reference.
With respect to the CYTb5, several other models of the role of this protein in P450 expression have been proposed besides its role as an electron carrier. Another model of the role of CYTb5 in P450 expression is based upon effects of protein-protein interactions as described, e.g., in Tamburini et al., Proc. Natl. Acad Sci., 84:11-15 (1986), incorporated herein by reference. By this model, CYTb5 binding with P450 results in a complex with increased high spin content. This complex then has a higher affinity for CPR. Through this interaction, CYTb5 does not actually provide the second electron, but decreases the time between first and second electron transfer. These conclusions were made based on work with rabbit CYP2B4. In addition, CYTb5 did not prevent interaction of rabbit CYP2B4 with CPR; therefore, it was proposed that the P450 binding domains for CYTb5 and CPR were different. This could possibly explain why only certain P450 interact with CYTb5. It is known that CYTb5 can be reduced by cytochrome b5 reductase or CPR as described, e.g., in Enoch et al., J. Bio. Chem, 254:8976-8981, (1979), incorporated herein by reference. However, in at least one interesting case, cytochrome b5 reductase addition to a reconstituted system containing CYTb5 did not result in increased substrate oxidation as described, e.g., in Sugiyama et al., J. Biochem., 87:1457-1467, (1979), incorporated herein by reference.
Various other studies have also indicated that CYTb5 may regulate phosphorylation of cytochrome P450""s by inhibiting cytochrome P450 phosphorylation by various protein kinases, e.g., cAMP dependent protein kinase and protein kinase C as described, e.g., in Jansson et al., Arch. Biochem. Biophys. 259:441-448 (1987), Epstein et al., Arch. Biochem. Biophys. 271(2):424-432(1989) and Lobanov et al., Biokhimiia 58(10):1529-1537 (1993).
Regardless of the mechanism, numerous studies indicate that CYTb5""s involvement is advantageous in some P450 mediated reactions. An obligatory role of CYTb5 has been shown in several reconstituted P450 systems as described, e.g., in Sugiyama et al., supra, Sugiyama et al. Biochem. and Biophys. Res. Comm., 90:715-720 (1979), Canova-Davis et al., J. Bio. Chem. 259:2541-2546, 1983, Kuwahara et al., Biochem. and Biophys. Res. Comm., 96:1562-1568 (1980), and Sasame et al., Life Sciences 14:35-46 (1974), each incorporated herein by reference. In all these cases, activity was abolished either upon omission of the exogenously added CYTb5 or its inactivation by the addition of CYTb5 antibody. As noted above, CYTb5 is also involved in the pathway for the second electron in certain P450 mediated reactions as described, e.g., in Hrycay et al., Archv. Bioch. 165:331-339 (1974), Imai et al., Biochem. and Biophys. Res. Comm. 75:420-426 (1977), and Imai, J. Biochem. 89:351-362 (1980), each incorporated herein by reference, as well as the reduction of oxy-cytochrome P450 to the active oxygen complex as described, e.g., in Noshiro et al., J. Biochem. 116:521-526 (1981), incorporated herein by reference.
In other studies using reconstituted systems, exogenously added CYTb5 increased the activities of rabbit CYP2B4 as described, e.g., in Sugiyama et al., J. Biochem., 92:1793-1803 (1982), and Chiang, Archv. Bioch. Biophy., 211:662-673 (1981), rabbit CYP1A2 as described, e.g., in Vatsis et al., J. Biol. Chem. 257:11221-11229 (1982), dog CYP2B11 and rat CYP2B1 as described, e.g., in Duignan et al., Arch. Bioch. Biophy. 267:294-304 (1988), and a rat P450 chlorobenzene hydroxylation reaction as described, e.g., in Lu et al., Biochem. and Biophys. Res. Comm. 61:1348:1355 (1974), each incorporated herein by reference.
CYP51, lanosterol 14xcex1 demethylase, performs an essential reaction in the biosynthesis of ergosterol, the major membrane sterol of Saccharomyces cerevisiae (Sc) as described, e.g., in Parks, CRC Crit. Rev. Microbiol., 6:301-341 (1978), incorporated herein by reference. Sc strains that synthesize ergosterol do not utilize exogenous ergosterol, but those that are blocked in pre-sterol steps or in heme biosynthesis can take up and use exogenous ergosterol as described, e.g., in Parks, supra. Anaerobically grown Sc cannot synthesize sterols but do utilize exogenous ergosterol. Disruption of CYP51 produces Sc strains which continue to produce 14xcex1-methyl sterols, produce no detectable ergosterol and are incapable of aerobic growth as described, e.g., in Kalb et al., DNA, 6:529-537 (1987), incorporated herein by reference. Rather they are obligate anaerobes, a condition where they do accumulate exogenous ergosterol.
CPR functions as the electron donor for this demethylase reaction as described, e.g., in Aoyama et al., J. Biol. Chem. 259:1661-1666 (1984), incorporated herein by reference. If CPR is the sole donor of electrons to CYP51 in Sc, CPR null mutants (cpr1) should phenotypically resemble the disrupted CYP51 strains (cyp51). However, a Sc cpr1 null mutant was not obligately anaerobic, and produced ergosterol as described, e.g., in Sutter and Loper, Biochem. and Biophys. Res. Comm., 160:1257-1266 (1989), incorporated herein by reference. The production of ergosterol shows that some CYP51 is still functional. The gene responsible for this recovery of a cpr1 null mutant was shown to be that encoding CYTb5, showing that in this system, CYTb5 is able to functionally mimic CPR when present in high copy number as described, e.g., in Truan et al., Gene, 142(1):123-7 (1994), incorporated herein by reference.
The expression of mammalian P450s in Sc has been accomplished with varied results as described, e.g., in Renaud et al., J. Biochem. 194:889-896 (1990), Urban et al., Biochimie 72:463-472 (1990), and Pompon et al., Molecular Endocrinology 3:1477-1487 (1989), each incorporated herein by reference. Studies have shown that activity by various mammalian P450s expressed in Sc has been enhanced upon addition of rabbit CYTb5 to isolated microsomes from these strains. Specifically, Sc microsomal human CYP3A4 and mouse Cyp1a-1 activity increased upon rabbit CYTb5 addition as described, e.g., in Renaud et al., supra, and Urban et al., supra.
In addition to its positive effects, one study involving Sc suggests CYTb5 may act negatively as described, e.g., in Pompon et al., supra. Addition of rabbit CYTb5 to isolated microsomes from a Sc expressing human CYP19 resulted in a decrease in aromatase activity.
Short chain (xe2x89xa6C12) aliphatic dicarboxylic acids (diacids) are important industrial intermediates in the manufacture of diesters and polymers, and find application as thermoplastics, plasticizing agents, lubricants, hydraulic fluids, agricultural chemicals, pharmaceuticals, dyes, surfactants, and adhesives. The high price and limited availability of short chain diacids are due to constraints imposed by the existing chemical synthesis.
Long-chain-diacids (aliphatic xcex1, xcfx89-dicarboxylic acids with carbon numbers of 12 or greater, hereafter also referred to as diacids) (HOOCxe2x80x94(CH2)nxe2x80x94COOH) are a versatile family of chemicals with demonstrated and potential utility in a variety of chemical products including plastics, adhesives, and fragrances. Unfortunately, the full market potential of diacids has not been realized because chemical processes produce only a limited range of these materials at a relatively high price. In addition, chemical processes for the production of diacids have a number of limitations and disadvantages. All the chemical processes are restricted to the production of diacids of specific carbon chain lengths. For example, the dodecanedioic acid process starts with butadiene. The resulting product diacids are limited to multiples of four-carbon lengths and, in practice, only dodecanedioic acid is made. The dodecanedioic process is based on nonrenewable petrochemical feedstocks. The multireaction conversion process produces unwanted byproducts, which result in yield losses, NOx pollution and heavy metal wastes.
Long-chain diacids offer potential advantages over shorter chain diacids, but their high selling price and limited commercial availability prevent widespread growth in many of these applications. Biocatalysis offers an innovative way to overcome these limitations with a process that produces a wide range of diacid products from renewable feedstocks. However, there is no commercially viable bioprocess to produce long chain diacids from renewable resources.
In accordance with the present invention, isolated nucleic acid encoding the CYTb5 protein (SEQ. ID. NO. 2) is provided. Also provided is an isolated CYTb5 protein including the amino acid sequence shown in SEQ. ID. NO. 2. Further provided is an expression vector including nucleic acid encoding the CYTb5 protein (SEQ. ID. NO. 2). Also provided is a host cell transformed with an expression vector comprising nucleic acid encoding the CYTb5 protein (SEQ. ID. NO. 2).
A method is provided of producing a CYTb5 protein including the amino acid sequence set forth in SEQ. ID. NO. 2 which includes transforming a host cell with a nucleic acid sequence that encodes the CYTb5 protein and culturing the cell in an appropriate medium.
A method of increasing the production of dicarboxylic acid is provided which includes providing a host cell having a naturally occurring number of CYTb5 genes; increasing, in the host cell, the number CYTb5 genes which encode a CYTb5 protein having the amino acid sequence set forth in SEQ. ID. NO. 2; and culturing the host cell in media containing an organic substrate which upregulates the CYTb5 gene, to effect increased production of dicarboxylic acid.